Mitochondrial activity and glutathione injury in apoptosis induced by unpreserved and preserved beta-blockers on Chang conjunctival cells

PURPOSE. Quaternary ammonium ions have been demonstrated to induce apoptosi s correlated with superoxide anion production in vitro. The purpose of this study was to further investigate the mechanisms of benzalkonium chloride ( BAC), unpreserved and preserved beta -blocker eye-drops-induced programmed cell death, with special attention to the roles of mitochondrial transmembr ane potential and intracellular reduced glutathione. METHODS. Chang conjunctival cells were incubated with different concentrati ons of unpreserved or preserved timolol (0.1%, 0.25%, and 0.4%), or carteol ol (1% and 2%), or BAC (0.0001% to 0.01%) for 15 minutes, or for 15 minutes with a 24-hour recovery period in normal medium. Cellular viability (neutr al red test), mitochondrial activity (rhodamine 123 test), intracellular re duced glutathione (monochlorobimane test), DNA condensation (Hoechst 33342 test), and reactive oxygen species (ROS) production (dichlorofluorescein di acetate and hydroethidine tests) were evaluated using microplate cold-light cytofluorometry. RESULTS. A significant, concentration-dependent decrease in cellular viabil ity was found with preserved beta -blockers and with BAC alone, whereas unp reserved preparations did not show any toxicity. Only preserved beta -block ers induced chromatin condensation associated with an alteration of mitocho ndrial activity and a decrease of glutathione, suggesting an apoptotic phen omenon. BAC increased glutathione after 15 minutes, whereas a decrease was observed after a recovery period. ROS production was found with preserved f ormulations at significantly higher levels than those observed with unprese rved drugs. CONCLUSIONS. This in vitro study demonstrates that oxidative stress, eviden ced by enhanced ROS production and mitochondrial. injury rather than by cel lular glutathione depletion, is a mechanism involved in apoptosis induced b y preservative containing eye-drops.